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autoBUSTER Documentation : Examples

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Contents


Normal refinement

To do a normal refinement only a PDB and MTZ file are needed:

% refine -p some.pdb -m other.mtz -d Results.1

Results available

The results of an autoBUSTER refinement (in the current directory or in the subdirectory pointed to with the "-d" flag) include:

Handling of waters

By default, the water structure will not be updated. This might be a good idea at a stage when the protein model has been built and refined and is very close to the final structure. At early stages of refinement (when the macro-molecule is still requiring major manual or automatic rebuilding), the placement of water molecules might not be ideal. On the other hand: if larger parts of the model are still missing, placing these so-called "waters" might indicate to the bulk solvent correction a much better and more realistic envelope. Similarly, towards the end of refinement - when water molecules have been checked manually - this feature should probably be left switched off.

Rigid-body refinement

When the starting model is poor or the cell parameters have changed (e.g. between an apo structure and a compound soak) it is a good idea to first start with some rigid-body refinement. This allows for collective motions that would otherwise take a lot of time or be impossible to achieve within a normal refinement.

NCS restraints

The recommended way of defining NCS is to start from the initial hypotheses that all copies of the macro-molecule within the asymmetric unit are identical. Only if there are clear indications that parts of one monomer differ from the rest (side-chains in crystal contacts, domain and loop movements, etc) should these parts be taken out of the NCS restraints. Therefore, the procedure to define NCS restraints should start from a completely restrained description that changes during the course of refinement and rebuilding to leave parts of the the molecules out. However, the final NCS restraints should probably still cover between 80-90 % of the atoms in each monomer.

The easiest way to define NCS restraints is using the -autoncs command-line flag. This will apply LSSR-type NCS restraints between all matching chains. It will automatically take care of real differences by removing those from the NCS-relation (so-called "pruning"). If the NCS-relation within the starting structure has been allowed to diverge too much (by over-eager model building into noisy maps or too agressive refinements), it might be a good idea to try and re-instate the NCS-relation. For that the pruning option can be switched off with -autoncs_noprune. This might also be necessary for situations where the X-ray data is rather weak, e.g. at lower resolution. But it depends a lot on the particular problem and especially the modeling history (NCS restraints are not something happening only during refinement, the manual model building also needs to be done under NCS restraints).

Another useful tool is the -sim_swap_equiv flag: this will try and correct problems where NCS-related atoms are chemically identical but have been given different atom names in the PDB files.

B-factor refinement

Under normal circumstances, the mode of B-factor refinement is determined automatically, depending on the resolution. At lower than 3.5 Å resolution the default is to turn off any B-factor refinement, whereas individual atomic B-factors are refined at higher than 3.5 Å.

Previous versions of autobuster used grouped B-factor models at moderate resolution (2.8 - 3.0 Å). However, we have found that with the use of tight BCORREL restraints (as implemented as default in BUSTER), use of individual B-factors gives superior results.

Individual B-factor refinement at lower than 3.5 Å resolution, or turning off B-factor refinement at higher than 3.5 Å, can be enforced by use of -B individual or -B None.

The resolution cutoff between these two schemes can be set with the parameter UseBrefNoneFrom.

More complex B-factor refinement modes can be set by use of the -B user option, in conjunction with -Gelly <gelly.file>. As an example, the following command may be used to refine a structure, defining a single B-factor per protein chain.

% refine -p some.pdb -m other.mtz -B user -Gelly gelly.dat

The gelly.dat file uses gelly combine syntax.

NOTE BUSTER_COMBINE B { A|* }
NOTE BUSTER_COMBINE B { B|* }

TLS refinement

To enable the use of TLS parametrisation, use the -TLS option of the refine command.

In its simplest invocation use:

% refine -p some.pdb -m other.mtz -TLS -d Results.1

This will perform TLS refinement for the first big cycle and do regular refinement for subsequent big cycles. If TLS definitions are present in the input pdb file header (both group definitions AND tensors), they will be used. Otherwise, it will define a single TLS group per macro-molecular chain.

Alternatively, use of:

% refine -p some.pdb -m other.mtz -TLS tls.dat -d Results.1

will similarly do TLS refinement for the first big cycle, but using TLS domain definitions specified in tls.dat (see TLS description for format details).

For convenience two different macros can be used.

NOTE: Any atoms that are not included in a TLS domain definition will undergo normal restrained refinement.

For a more detailed description of the use of these TLS options please see the TLS tutorial WIKI.


Some ligand is (possibly) present, but location is not well known

The -L flag tells the program to remove water atoms around residual difference density at the last cycle. This should make the difference density in these (potentially) 'interesting' regions clearer. The starting PDB file should obviously not contain any atoms for the unknown ligand.

% refine -p some.pdb -m other.mtz -L -d Results.2

The file Results.2/analyse.html can be used to look at pictures of the found (possible) binding sites.


A ligand is (possibly) present, and the location is well known

If the location of the binding site of a new ligand is known (e.g. from previously solved structures, biochemical data or docking experiments), a PDB file with a model of this (or a similar) ligand can be given with the -Lpdb flag. This PDB file should not contain the putative ligand as present in the crystal or even a similar structure (the risk of introducing bad model bias would be unacceptably high), but just a collection of atoms that cover the space likely to be occupied by the unknown ligand structure, without highlighting its shape.

This option tells the program to remove waters atoms around this PDB file at the last cycle. This should make the difference density in these 'interesting' regions clearer.

Note :  Be careful, when using dummy atoms to describe a large area in space: these atoms are also used to describe the region not covered by bulk solvent. So if these dummy atoms are within the bulk solvent region, some artificial difference density will appear (corresponding to the bulk solvent).

% refine -p some.pdb -m other.mtz -Lpdb lig-model.pdb -d Results.3

The file Results.3/analyse.html can be used to look at pictures of densities within the user-defined binding sites.


A ligand is (possibly present) in a known location. A variation: excluding regions from bulk solvent during refinement

Use the -x flag to exclude a region described by the provided PDB file from both water addition and bulk solvent region throughout the refinement. This should make the difference density in this region clearer.

However, there is always the danger of creating a biased imprint of the used PDB file in cases where nothing has bound in that site. Under those circumstances, the difference density visible is due to unmodelled bulk solvent (since the region is left out of the bulk-solvent mask). Be careful when decreasing the density level while looking at maps, especially Fo-Fc difference density maps: if one has to go to a level at which there is a lot of difference density all over the remainder of the model, it is unlikely to be significant.


Some settings that might need adjustment

Here are some flags that might need changing:
Last modification: 14.03.2016